The proposed research is directed at demonstration of a protein surface mapping technique based on novel chemical labeling methods that can be combined with high resolution mass spectrometric characterization to identify surface accessible amino acids residues in native-folded proteins. This information then will be utilized in an integrated fashion with computational structural prediction methods to enhance their accuracies and throughput. If successful, this method could enhance dramatically the structural characterization throughput (albeit at moderate resolution) of a wide range of proteins, and provide critical input into the refinement of computational prediction methods. To achieve this goal, four specific aims are proposed. Specific Aim 1 focuses on formulation and characterization of an experimental surface mapping protocol that includes a toolbox of labeling reagents for protein structural determinations. We propose to optimize our radical labeling approach by defining the experimental parameters for quantitative labeling, background reduction, and alternate reagent development. The goal of this task will be to develop an experimental toolbox for labeling that includes a variety of reagents. Specific Aim 2 is directed toward demonstration of the surface mapping technique for large proteins and protein mixtures, two areas that are difficult for XRC and NMR techniques to examine. Specific Aim 3 involves demonstration of the surface mapping technique for characterizing protein conformational changes, to illustrate how this experimental approach can provide more than only low resolution structural information. Specific Aim 4 seeks to integrate surface mapping data as experimental constraints for computational protein structural prediction, involving both protein threading algorithms (PROSPECT) and ab initio methods (Rosetta). One favorable outcome if the proposed experimental approach is successful is the large amount of structural data at moderate resolution that can be generated from protein mixtures. At present, this experimental capability is non-existent.